These studies will investigate the molecular mechanisms by which PTH and PTHrP ligands bind to and activate the PTH/PTHrP receptor (PTHR1): The PTHR1 plays vital roles in calcium and phosphate homeostasis, and in bone growth and remodeling. We have new data to suggest that the PTHR1 can adopt different conformations, and that different PTH and PTHrP ligands bind to these conformations with different selectivities, and thus induce different biological responses. Thsu, PTH(1-34) and certain other ligands, but not PTHrP(1-36), bind with high affinity to a novel conformation, called RO, which is insensitive to GTPgammaS, and thus, are not likely coupled to G proteins. Ligands that bind preferentially to RO produce prolonged signaling responses in cells and in animals. We thus hypothesize that RO is a stable intermediary that can convert to active-state, RG. This pedicts that RO-selective ligands will produce prolonged signaling. In contrast, RG-selective ligands will produce short-lived, pulsatile, signaling responses. These hypotheses have important implications, not only for understanding the fundamental mechanisms by which the PTHR1 mediates the biological actions of PTH and PTHrP, endocrine and paracrine, respectively, but also for developing new PTHR1-based therapies for diseases such as osteoporosis and hypoparathyroidism, for which signal duration time appears critical. Our goal is to elucidate the underlying biochemical and cellular mechanisms involved, and to confirm the hypothesis that conformational selectivity at the PTHR1 is biologically relevant, and indeed governs the actions of PTH and PTHrP ligands in vivo. To do this, we will use an integrated experimental design approach that incorporates molecular and pharmacological methods, FRET-based biophysical methods, high-resolution sub-cellular imaging methods, and in vivo mouse model systems. As confirmation of our hypotheses, we will develop and test new PTHR1 ligands that have enhanced conformational selectivity, and thus altered biological and signaling actions. In particular, we will develop new long-acting RO-selective ligands, and assess whether they are superior to PTH(1-34) in normalizing calcium in models of hypoparathyroidism. We also will develop short-acting, RG-selective ligands, and assess whether they have potent anabolic effects on bone with minimal resorptive/calcemic effects. Such agents could represent the next generation of PTHR1 ligands for treating osteoporosis.